ABSTRACT Proteotoxic stress or inhibition of cellular proteasome activity by proteasome inhibitor drugs sets in motion an evolutionarily conserved pathway that directs the de novo synthesis of proteasomes as a compensatory response. Our previous studies established the transcription factor Nrf1 as a key player in this stress-response pathway. Nrf1, by its ability to bind to the anti-oxidant response elements typically found in the regulatory regions of proteasome genes, induces their expression in response to proteasome inhibition. As an endoplasmic reticulum (ER)-bound transcription factor with a bulk of its polypeptide in the lumen, Nrf1 activation involves its retrotranslocation into the cytosol in a manner that depends on the ATPase p97/VCP. This is followed by proteolytic processing and subsequent mobilization of the transcriptionally active form of Nrf1 to the nucleus. Further understanding of the Nrf1 pathway could shed light on the intricate mechanisms by which cells cope with proteotoxic stress. In the first two aims, we propose to dissect the functional output and the mechanism of activation of Nrf1 pathway and explore various factors that assist in mobilizing this transcription factor from the lumen of the ER all the way to the nucleus. Using the information gleaned from the above two aims and using genetic and chemical tools, in the third aim, we propose to test if inhibition of Nrf1 pathway leads to increased efficacy of proteasome inhibitor treatment in cancer cells. Thus, the proposed line of work is important not only from a basic research stand-point of furthering Nrf1 biology; it is also significant from a translational perspective as well, since it has the potential to illuminate novel strategies to modulate the cellular protein clearance pathways in various human diseases.